Novel Process for the Preparation of Hexacyclic Compounds

ABSTRACT

This invention relates to novel processes for the preparation of compounds of the formula [1], 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3  and R 4  are as defined in the claims and description as well as pharmaceutically acceptable salts thereof. The compounds of the formula [1] are hexacyclic compounds having potent anti-tumor activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/546,287, filed on Mar. 20, 2006, the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/JP2004/002023, which has anInternational filing date of Feb. 20, 2004, designated the United Statesof America and claims the benefit of European Patent Application SerialNo. 03003232.0, filed on Feb. 21, 2003, each of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a novel process for the preparation ofnovel hexacyclic compounds (camptothecin analogs) having potentanti-tumor activity.

BACKGROUND ART

The cytotoxic activity of camptothecin is attributable to its ability tointerfere with DNA topoisomerase I (Hsiang, Y.-H. et al. Camptothecininduces protein-linked DNA breaks via mammalian DNA topoisomerase I. J.Biol. Chem., 260:14873-14878, 1985). DNA topoisomerase I is aphosphorylated protein and is required for DNA replication,transcription and recombination. It forms a covalent reversible DNAtopoisomerase I-double strand DNA complex (referred to as cleavablecomplex) and relaxes supercoiled DNA by cleaving and religating one ofthe two DNA strands (see for example Wang, J. C. DNA topoisomerases.Annu. Rev. Biochem. 54:665-697, 1985; Champoux, J. J. Mechanisticaspects of type-I topoisomerase. In “DNA topology and its biologicaleffects” pp. 217-242, 1990). Camptothecin reversibly interacts with thecleavable complex and subsequently induces DNA single strand breaks byinterfering with the religation step (Hsiang, Y.-H. et al. Camptothecininduces protein-linked DNA breaks via mammalian DNA topoisomerase I. J.Biol. Chem., 260:14873-14878, 1985; Porter, S. E. et al. The basis forcamptothecin enhancement of DNA breakage by eukaryotic DNA topoisomeraseI. Nucleic Acid Res. 17:8521-8532, 1989).

Although DNA topoisomerase I is an ubiquitous enzyme and is presentthroughout the cell cycle, antiproliferative activities of camptothecinare only limited in clinical trials, and half-life in plasma ofcamptothecin appeared to be short (less than 30 min) being converted tothe inactive carboxylate form. Furthermore, camptothecin is poorlysoluble in water, and therefore, it itself can not be formulated for theuse of intravenous injection.

A number of camptothecin derivatives were synthesized to improveanti-tumor activity, lactone stability in plasma and/or watersolubility, and were tested clinically (Gerrits, C. J. H., de Jonge, M.J. et al. Topoisomerase I inhibitors: the relevance of prolongedexposure for clinical development. Br. J. Cancer, 76: 952-962, 1997;O'Leary, J. et al. Camptothecins: a review of their development andschedules of administration. Eur. J. Cancer, 34: 1500-1508, 1988;Gerderblom, H. A. et al. Oral topoisomerase I inhibitors in adultspatients: present and future. Investig. New Drugs, 17: 401-415, 1999).

However, at the present time, only two camptothecin derivatives,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycatnptothecin(irinotecan) that is the prodrug of 7-ethyl-10-hydroxycamptothecin(SN-38, EP 0074256) and 9-(dimethylamino)methyl-10-hydroxycamptothecin(topotecan) have been introduced into clinical practice (Kunimoto, T. etal. Antitumor activity of7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin, anovel water-soluble derivative of camptothecin, against murine tumors.Cancer Res., 47:5944-5947, 1987; Kingsbury, W. D. et al. Synthesis ofwater-soluble (aminoalkyl)camptothecin analogs: inhibition oftopoisomerase I and antitumor activity. J. Med. Chem., 34:98-107, 1991).

Due to the structural complexity of camptothecin, there is clearly alimitation of derivatization of camptothecin and synthetic routespreparing them. Thus, there are still strong needs to discover newsynthetic routes for delivering new camptothecin analogs with improvedactivities.

DISCLOSURE OF THE INVENTION

It has been shown that the novel hexacyclic compounds represented by thefollowing formula [1],

wherein

-   R¹ and R² are independently hydrogen, halogen, (C₁-C₈) alkyl or    (C₁-C₅) alkoxy;-   R³ is hydrogen;    -   (C₁-C₅)-alkyl optionally substituted with one to three moieties        independently selected from the group consisting of        (C₁-C₅)-alkoxy, hydroxy, halogen, amino, (C₃-C₇)-cycloalkyl,        heterocyclic ring or    -   aryl in which the aryl ring is optionally substituted with one        to three moieties independently selected from the group        consisting of hydroxy, (C₁-C₅)-alkoxy, amino,        mono-(C₁-C₅)-alkylamino or di-(C₁-C₅)-alkylamino;-   R⁴ is hydrogen;    -   (C₁-C₁₀)-alkyl, optionally substituted with one to three        moieties independently selected from the group consisting of        (C₁-C₅)-alkoxy, hydroxy, halogen, amino,        mono-(C₁-C₅)-alkylamino, di-(C₁-C₅)-alkylamino and        (C₃-C₇)-cycloalkyl, a heterocyclic ring and aryl in which the        aryl ring is optionally substituted with one to three moieties        independently selected from the group consisting of hydroxy,        alkoxy, halogen, amino, mono-(C₁-C₅)-alkylamino and        di-(C₁-C₅)-alkylamino;        and pharmaceutically acceptable salts thereof,        exhibit potent anti-tumor activities during studies using human        tumor xenograft models.

The compounds of the formula [1] can be prepared from9-nitrocamptothecin derivatives as illustrated in the following Scheme1.

However, those processes are accompanied with some disadvantages, forexample, long linear synthetic steps, low overall yields and manychromatographic purification steps, which set tremendous challenge forthe production in industrial scale.

Therefore it is an object of the present invention to provide a noveland more sufficient synthetic process for the preparation of hexacycliccompounds of the formula [1],

wherein R¹, R², R³ and R⁴ are the same as defined above,

and pharmaceutically acceptable salts thereof,which comprises condensing a compound of the formula [2] or its salt,

wherein R¹, R², R³, R⁴ are the same as defined above and R⁵ is —OH or—O—(C₁-C₅)-alkyl,

with a compound of the formula [3],

wherein R⁶ is chlorine or bromine.

More specifically, it is another object of the present invention toprovide a process for the preparation of the compound of the formula[1A],

which comprises condensing the compound of the formula [2A] or its salt,

with a compound of the formula [3A].

It is another object of the present invention to provide a process forthe preparation of compounds of the formula [2],

wherein R¹, R², R³, R⁴ and R⁵ are the same as defined above,

which comprises

a) ozonolysis of compounds of the formula [4] or salts thereof,

wherein R¹, R² and R³ are the same as defined above,

in order to obtain compounds of the formula [5],

wherein R¹, R² and R³ are the same as defined above,

b) cyclizing the compounds of the formula [5] with amines R⁴—NH₂ (R⁴ isthe same as defined above) in order to obtain compounds of the formula[6],

wherein R¹, R², R³, R⁴ and R⁵ are the same as defined above,

c) hydrogenating the nitro group of compounds of the formula [6] inorder to obtain the compound of the formula [2],

wherein R¹, R², R³, R⁴ and 15 are the same as defined above.

Another object of the present invention is to provide a process for thepreparation of compounds of the formula [2] which, after ozonolysis ofcompounds of the formula [4] giving compounds of the formula [5],comprises,

d) hydrogenating the nitro group of compounds of the formula [5],

-   -   wherein R¹, R² and R³ are the same as defined above,

in order to obtain compounds of the formula [7],

wherein R¹, R² and R³ are the same as defined above,

e) cyclizing compounds of the formula [7] with amines R⁴—NH₂, wherein R⁴is the same as defined above, in order to obtain compounds of theformula [2].

More specifically, it is another object of the present invention toprovide a process for the preparation of the compound of formula [2A]giving the compound of the formula [5A],

which comprises

a′) ozonolysis of a compound of the formula [4A] or its salt,

in order to obtain the compound of the formula [5A],

to b′) cyclizing the compound of the formula [5A] with n-pentylamine inorder to obtain the compound of the formula [6A],

c′) hydrogenating the nitro group of compound of the formula [6A] inorder to obtain the compound of the formula [2A].

Another object of the present invention is to provide a process for thepreparation of the compound of the formula [2A] which, after ozonolysisof the compound of the formula [4A] giving the compound of the formula[5A], comprises

d′) hydrogenating the nitro group of the compound of the formula [5A],

in order to obtain the compound of the formula [7A],

e′) cyclizing the compound of the formula [7A] with n-pentylamine inorder to obtain the compound of the formula [2A].

It is another object of the present invention to provide a process forthe preparation of the compounds of the formula [3], which comprises

halogenating the compound of the formula [8]

in order to obtain a compound of formula [3],

wherein R⁶ is chlorine or bromine.

The above mentioned process can be summarized in to the followingreaction scheme 2.

Unless otherwise indicated, the following definitions are set forth toillustrate and to define the meaning and scope of the various terms usedto describe the invention herein.

The term “alkyl” refers a straight or branched monovalent saturatedaliphatic hydrocarbon group.

“(C₁-C₁₀)-alkyl” means a straight chain or branched hydrocarbon chainhaving 1 to 10, preferably 1 to 8 carbon atom(s), e.g. methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl,1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl,2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl,2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,2-dimethylbutyl,2,3-dimethylbutyl, 3,3-dimethylbutyl and the like, more preferablybutyl, isobutyl, 3-methylbutyl, or pentyl.

“(C₁-C₅)-alkyl” preferably means methyl, ethyl, propyl, isopropyl,butyl, isobutyl, ter-butyl, sec-butyl, pentyl, 1-methylbutyl,2-methylbutyl, 3-methylbutyl and the like, more preferably methyl,ethyl, propyl or isopropyl.

The term “alkoxy” refers to the group —O—R′, wherein R′ is an alkylgroup as defined above. “(C₁-C₅)-alkoxy” preferably means methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy,3-methylbutoxy, 2,2-dimethylpropoxy and the like.

The term “hydroxy” refers to the group HO—.

The term “halogen” refers to fluoro, chloro, bromo and iodo.

The term “amino” refers to the group —NH₂ and includes amino groupswhich are protected by a group known in the art such as a formyl,acetyl, trithyl, tert-butoxycarbonyl, benzyl, benzyloxycarbonyl, and thelike. Preferably, it means —NH₂.

The term “mono-alkylamino” refers to the group —NH—R′, wherein R′ is analkyl group as defined above, and includes amino groups which areprotected by a group known in the art such as a formyl, acetyl, trityl,tert-butoxycarbonyl, benzyl, benzyloxycarbonyl, and the like. The term“mono-(C₁-C₅)-alkylamino” preferably means N-methylamino, N-ethylamino,N-propylamino, N-isopropylamino, N-butylamino, N-(1-methylpropyl)-amino,N-(2-methylpropyl)amino, N-pentylamino, and the like, more preferablyN-ethylamino, N-propylamino, or N-butylamino.

The term “di-alkylamino” refers to the group —NR′R″, wherein R′ and R″are (independently from each other) an alkyl group as defined above.“di-(C₁-C₅)-alkylamino” preferably means N,N-dimethylamino,N,N-diethylamino, N,N-dipropylamino, N,N-diisopropylamino,N,N-dibutylamino, N-methyl-N-ethylamino, N-methyl-N-propylamino and thelike, more preferably N,N-dimethylamino, or N,N-diethylamino.

The term “(C₃-C₇)-cycloalkyl” means 3 to 7 membered ring, which do notcontain any heteroatoms in the ring. “Cycloalkyl” preferably meanscyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and thelike, more preferably cyclopentyl and cyclohexyl.

The term “heterocyclic ring” refers a 3 to 10 membered heterocyclyl orheteroaryl ring which contains one or more heteroatom(s) selected fromN, S and O. Preferably the heterocyclic ring is selected from the groupconsisting of oxazolyl, thiazolyl, 4,5-dihydro-oxazolyl,4,5-dihydro-thiazolyl, furyl, pyrrolyl, thienyl, imidazolyl, triazolyl,tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, triazinyl, oxadiazolyl,thiadiazolyl, pyrrolidinyl, tetrahydrothienyl, tetrahydrofuryl,morpholinyl, piperidyl, piperazinyl, 1-methylpiperazinyl and the like,more preferably imidazolyl, pyridyl, morpholinyl and pyrrolidinyl.

The term “aryl” means an aromatic carbocyclic group, i.e. a 6 or 10membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl ortetrahydronaphthyl, preferably phenyl or naphthyl, and most preferablyphenyl.

In the present invention, the expression “optionally substituted with”means that substitution can occur at one to three positions, preferablyat one position, and, unless otherwise indicated, that the substituentsare independently selected from the specified options.

The term “pharmaceutically acceptable,” such as pharmaceuticallyacceptable carrier, excipient, prodrug, etc., means pharmacologicallyacceptable and substantially non-toxic to the subject to which theparticular compound is administered.

The term “pharmaceutically acceptable salt” refers to conventionalacid-addition salts or base-addition salts which retain the biologicaleffectiveness and properties of the hexacyclic compounds of the formula[1] and are formed from suitable non-toxic organic or inorganic acids ororganic or inorganic bases. The acid-addition salts include thosederived from inorganic acids such as hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid andnitric acid, and those derived from organic acids such asp-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalicacid, succinic acid, citric acid, malic acid, lactic acid, fumaric acidand the like. The base-addition salts include those derived frompotassium, sodium, ammonium, and quartemary ammonium hydroxide, such asfor example tetramethylammonium hydroxide.

In the above definitions, the preferable embodiments of R³ are hydrogen,methyl, ethyl, propyl, isopropyl, butyl, pentyl, hydroxymethyl,methoxymethyl, acetoxymethyl, aminomethyl, (methylamino)methyl,(dimethylamino)methyl, fluoromethyl, chloromethyl, trifluoromethyl,phenyl, pyridin-2-yl, methoxy, ethoxy, methylthio, ethylthio, amino,methylamino, ethylamino, propylamino, butylamino, dimethylamino,diethylamino and the like, and more preferably hydrogen, methyl, ethyl,propyl, hydroxymethyl, aminomethyl, chloromethyl, trifluoromethyl.

The preferable embodiments of R⁴ are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, pentyl, 3-methylbutyl, 2,2-dimethylpropyl,1-ethylpropyl, hexyl, 4-methylpentyl, 3,3-dimethylbutyl, heptyl,2-methoxyethyl, 2-ethoxyethyl, 2-propoxylethyl, 3-methoxypropyl,4-methoxybutyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl,5-hydroxypentyl, 2-fluoroethyl, 3-fluoropropyl, 4-fluorobutyl,2-chloroethyl, 3-chloropropyl, 4-chlorobutyl, 2-(dimethylamino)ethyl,3-(dimethylamino)propyl, 2-(cyclohexyl)ethyl, 2-(4-morpholino)ethyl,2-(pyyrrolidino)ethyl, 2-(piperidino)ethyl, 2-(4-methylpiperazino)ethyl,2-(pyridin-2-yl)ethyl, 2-(pyridin-3-yl)ethyl, 2-(imidazol-1-yl)ethyl,benzyl, phenethyl, 2-(1-naphthyl)ethyl, 3-phenylpropyl,2-(4-fluorophenyl)ethyl, 2-(4-chlorophenyl)ethyl,2-(4-methoxyphenyl)ethyl, 2-(4-hydroxyphenyl)ethyl,2-[4-(dimethylamino)phenyl]ethyl, 2-(3,4-methyenedioxyphenyl)ethyl andthe like, more preferably methyl, ethyl, propyl, isopropyl, butyl,isobutyl, pentyl, 3-methylbutyl, hexyl, 3,3-dimethylbutyl, heptyl,2-hydroxyethyl, 3-hydroxypropyl, 2-(dimethylamino)ethyl,3-(dimethylamino)propyl, 2-(4-morpholino)ethyl, 2-(pyridin-2-yl)ethyl,2-(pyridin-3-yl)ethyl, benzyl, phenethyl, 3-phenylpropyl,2-(4-fluorophenyl)ethyl, 2-(4-chlorophenyl)ethyl,2-(4-methoxyphenyl)ethyl and the like.

In more detail, the present invention refers to a process for thepreparation of a compound of the formula [1],

wherein

-   R¹ and R² are independently hydrogen, halogen, (C₁-C₅)-alkyl or    (C₁-C₅)-alkoxy;

R³ is hydrogen;

-   -   (C₁-C₅)-alkyl optionally substituted with one to three moieties        independently selected from the group consisting of        (C₁-C₅)-alkoxy, hydroxy, halogen, amino, (C₃-C₇)-cycloalkyl,        heterocyclic ring or aryl in which the aryl ring is optionally        substituted with one to three moieties independently selected        from the group consisting of hydroxy, (C₁-C₅)-alkoxy, amino,        mono-(C₁-C₅)-alkylamino or di-(C₁-C₅)-alkylamino;

R⁴ is hydrogen;

-   -   (C₁-C₁₀)-alkyl, optionally substituted with one to three        moieties independently selected from the group consisting of        (C₁-C₅)-alkoxy, hydroxy, halogen, amino,        mono-(C₁-C₅)-alkylamino, di-(C₁-C₅)-alkylamino and        (C₃-C₇)-cycloalkyl, a heterocyclic ring or aryl in which the        aryl ring is optionally substituted with one to three moieties        independently selected from the group consisting of hydroxy,        alkoxy, halogen, amino, mono-(C₁-C₅)-alkylamino and        di-(C₁-C₅)-alkylamino;        and pharmaceutically acceptable salts thereof,

which comprises condensing a compound of the formula [2] or its salt,

wherein R¹, R², R³, R⁴ are the same as defined above and R⁵ is OH orO—(C₁-C₅)-alkyl,

with a compound of the formula [3] wherein R⁶ is a chlorine or bromine,

The condensation reaction of [2] and [3] to compound [1] is preferablycarried out in suitable solvent in the presence or absence of an acidcatalyst. Preferred solvents are methanol, ethanol, toluene,dichloromethane, chloroform, acetic acid and formic acid as like, mostpreferably acetic acid and formic acid. Preferred acid catalysts arehydrochloric acid, p-toluenesulfonic acid, and trifluoroacetic acid. Thereaction is preferably conducted at the temperature between roomtemperature to 150° C., more preferably between 50° C. to 100° C.

More preferably, the process of the present invention is for thepreparation of compounds of formula [1], wherein

-   R¹ is hydrogen; and-   R² is hydrogen or (C₁-C₃)-alkyl;-   R³ is hydrogen;    -   (C₁-C₅)-alkyl optionally substituted with one to three moieties        independently selected from the group consisting of        (C₁-C₅)-alkoxy, hydroxy, halogen, amino,    -   (C₃-C₇)-cycloalkyl, a heterocyclic ring and aryl; and-   R⁴ is hydrogen;    -   (C₁-C₈)-alkyl which is optionally substituted with one to three        moieties independently selected from the group consisting of        (C₁-C₃)-alkoxy, hydroxy, halogen, amino,        mono-(C₁-C₃)-alkylamino, di-(C₁-C₃)-alkylamino,        (C₃-C₇)-cycloalkyl, a heterocyclic ring and aryl in which the        aryl ring is optionally substituted with one to three moieties        independently selected from the group consisting of hydroxy,        alkoxy and halogen.

Most preferably, the process of the present invention concerns thepreparation of compounds of the formula [1], wherein

-   R¹ and R² are hydrogen;-   R³ is hydrogen, methyl, ethyl, propyl, hydroxymethyl, aminomethyl,    (methylamino)methyl, (dimethylamino)methyl, chloromethyl,    trifluoromethyl; and-   R⁴ is methyl, ethyl, n-propyl, 1-methylethyl, n-butyl,    1,1-dimethylethyl, 2-methylpropyl, 2,2-dimethylpropyl, n-pentyl,    3-methylbutyl, 2-n-hexyl, 3,3-dimethylbutyl, n-heptyl, n-octyl,    benzyl, phenethyl, 2-(dimethylamino)ethyl, 2-(4-morpholino)ethyl,    3-(dimethylamino)propyl, 2-(pyridin-2-yl)ethyl,    2-(pyridin-3-yl)ethyl, 2-(4-methoxyphenyl)ethyl,    2-(4-chlorophenyl)ethyl or 2-(4-fluorophenyl)ethyl, 3-phenylpropyl.

Examples for compounds of the formula [1] prepared by the process arecompounds selected from the group consisting of:

-   a) (9S)-1-butyl-9-ethyl-9-hydroxy-1H,    12H-pyrano[3″,4″:6′,7′]indolizino-[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   b) (9S)-9-ethyl-9-hydroxy-1-[2-(4-morpholino)ethyl]-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino-[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,15H)-dione    hydrochloride;-   c) (9S)-9-ethyl-9-hydroxy-1-propyl-1H,    12H-pyrano[3″,4″:6′,7′]indolizino[1′,2′:6,5]-pyrido-[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   d) (9S)-1-benzyl-9-ethyl-9-hydroxy-1H,    12H-pyrano[3″,4″:6′7′]indolizino-[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   e) (9S)-9-ethyl-9-hydroxy-1-phenethyl-1H,    12H-pyrano[3″,4″:6′7′]indolizino-[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   f) (9S)-2.9-diethyl-9-hydroxy-1-phenethyl-1H,    12H-pyrano[3″,4″:6′7′]indolizino-[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   g) (9S)-9-ethyl-9-hydroxy-1-(3-phenylpropyl)-1H,    12H-pyrano[3″,4″:6′7′]indolizino-[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   h) (9S)-9-ethyl-9-hydroxy-1-(3-methylbutyl)-1H,    12H-pyrano[3″,4″:6′7′]indolizino-[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   i) (9S)-2.9-diethyl-9-hydroxy-1-(3-methylbutyl)-1H,    12H-pyrano[3″,4″:6′7′]-indolizino-[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   j) (9S)-2.9-diethyl-9-hydroxy-1-(2-methylpropyl)-1H,    12H-pyrano[3″,4″:6′7′]-indolizino[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   k) (9S)-9-ethyl-1-heptyl-9-hydroxy-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[l′,    2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H, 15H)-dione;-   l) (9S)-9-ethyl-9-hydroxy-1-methyl-1H,    12H-pyrano[3″,4″:6′,7″]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   m) (9S)-9-ethyl-9-hydroxy-1-(2-methylpropyl)-1H,    12H-pyrano[3″,4″:6′,7′]indolizino-[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-110,13(9H,    15H)-dione;-   n) (9S)-9-ethyl-1-hexyl-9-hydroxy-1H,    12H-pyrano[3″,4″:6′,7′]indolizino-[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   o) (9S)-9-ethyl-9-hydroxy-1-pentyl-1H,    12H-pyrano[3″,4″:6′,7′]indolizino-[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   p) (9S)-1,9-diethyl-9-hydroxy-1H,    12H-pyrano[3″,4″:6′,7′]indolizino-[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   q) (9S)-9-ethyl-9-hydroxy-1-[2-(4-methoxyphenyl)ethyl]-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino-[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   r) (9S)-1-[2-(4-chlorophenyl)ethyl]-9-ethyl-9-hydroxy-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   s) (9S)-9-ethyl-1-[2-(4-fluorophenyl)ethyl]-9-hydroxy-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   t) (9S)-9-ethyl-1-[2-(4-fluorophenyl)ethyl]-9-hydroxy-2-methyl-1H,    12H-pyrano[3″,4″:6′,7′]indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13    (9H,15H)-dione;-   u) (9S)-9-ethyl-9-hydroxy-1-(1-methylethyl)-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]    quinazoline-10,13(9H, 15H)-dione;-   v)    (9S)-1-(3,3-dimethylbutyl)-9-ethyl-9-hydroxy-1H,12H-pyrano[3″,4″:6′,7′]-indolizino[1′,    2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H, 15H)-dione;-   w) (95)-2,9-diethyl-9-hydroxy-1-(3-methylbutyl)-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   x) (9S)-9-ethyl-9-hydroxy-1-(2-hydroxyethyl)-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   y) (9S)-9-ethyl-9-hydroxy-1-(2-hydroxyethyl)-2-methyl-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   z) (9S)-9-ethyl-9-hydroxy-2-methyl-1-pentyl-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   aa) (9S)-2,9-diethyl-9-hydroxy-1-pentyl-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   bb) (9S)-9-ethyl-9-hydroxy-1-pentyl-2-propyl-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   cc) (95)-9-ethyl-9-hydroxy-2-hydroxymethyl-1-pentyl-1H,    12H-pyrano[3″,4″:6′7′]-indolizino[1′,    2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H, 15H)-dione;-   dd) (9S)-9-ethyl-9-hydroxy-2-hydroxymethyl-1-(2-methylpropyl)-1H,    12H-pyrano-[3″,4″:6′7′]indolizino[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   ee) (9S)-9-ethyl-9-hydroxy-2-hydroxymethyl-1-(3-methylbutyl)-1H,    12H-pyrano[3″,4″:6′7′]indolizino[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   ff) (9S)-2-chloromethyl-9-ethyl-9-hydroxy-1-(3-methylbutyl)-1H,    12H-pyrano-[3″,4″:6′7′]indolizino[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione;-   gg) (9S)-2-aminomethyl-9-ethyl-9-hydroxy-1-pentyl-1H,    12H-pyrano[3″,4″:6′7′]-indolizino[1′2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione; and-   hh) (9S)-9-Ethyl-9-hydroxy-1-pentyl-2-trifluoromethyl-1H,    12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,    15H)-dione.

An especially preferred process of the present invention is that for thepreparation of compound of formula [1A],

which comprises condensing the compound of the formula [2A] or its salt,

with the compound of the formula [3A],

Another embodiment of the present invention concerns the process for thepreparation of compounds of the formula [2], which comprises

a) ozonolysis of the compound of the formula [4] or its salt,

wherein R¹, R² and R³ are the same as defined above,in order to obtain compound of the formula [5],

wherein R¹, R² and R³ are the same as defined above,

b) cyclizing the compound of formula [5] with an amine R⁴—NH₂ to obtaina compound of the formula [6],

wherein R¹, R², R³, R⁴ and R⁵ are the same as defined above,

c) hydrogenating the nitro group of compound of the formula [6] in orderto obtain a compound of the formula [2],

wherein R¹, R², R³, R⁴ and R⁵ are the same as defined above.

Alternatively, the present invention is to provide a process for thepreparation of a compound of the formula [2] which, after ozonolysis ofa compound of the formula [4] giving a compound of the formula [5],comprises

d) hydrogenating the nitro group of compound of the formula [5],

wherein R¹, R² and R³ are the same as defined above,in order to obtain a compound of the formula [7],

wherein R¹, R² and R³ are the same as defined above,

e) cyclizing the compound of the formula [7] with an amine R⁴—NH₂ inorder to obtain a compound of the formula [2],

wherein R¹, R², R³, R⁴ and R⁵ are the same as defined above.

The ozonolysis in the above step a) is preferably carried out in asuitable solvent. Suitable solvents are dichloromethane, chloroform,tetrahydrofuran, N,N-dimethyl-formamide, acetic acid, methanol and wateras like and a mixture of solvents indicated above, more preferablydichloromethane, dichloromethane-methanol mixture anddichiloromethane-N,N-ditnethylforamide mixture. Typically, theozonolysis is conducted at the temperature between −78° C. to roomtemperature for 1 to 10 hours. After completion of the reaction, thereaction mixture may be treated with a suitable reducing reagent.Preferable reducing reagents are dialkyl sulfide such as dimethylsulfide and diethyl sulfide and trialkyl- or triarylphosphines such astributylphosphine and triphenyl phosphine, most preferably dimethylsulfide.

The cyclization in the above step b) or step e) with an amine R⁴—NH₂ ispreferably carried out in a suitable organic solvent such as methanol,ethanol, tetrahydrofuran, 1,4-dioxane, chloroform, N,N-dimethylformamide as like, more preferably methanol and ethanol. The reactionwas conducted at the temperature from room temperature to 120° C. for aperiod of 15 minutes to several days.

The hydrogenation in the above step c) or step d) is preferably carriedout with a reducing reagent in a suitable solvent in the presence of asuitable catalyst. Suitable reducing reagents are molecular hydrogen, orhydrogen source such as cyclohexadiene, formic acid, ammonium formate aslike. Suitable solvents are e.g., methanol, ethanol, ethyl acetate,N,N-dimethyl formamide, 1,4-dioxane, and water with or without inorganicor organic acid such as 1 to 10 N aqueous hydrochloric acid, sulfonicacid, phosphonic acid, nitric acid, acetic acid, frifluoroacetic acid.Suitable catalysts are transition metals such as palladium, platinum,nickel, and typically are 5 to 20% palladium on charcoal and 5 to 10%palladium hydroxide on charcoal. Typically the reaction is conducted atthe temperature 0 to 100° C., at the pressure 1 to 100 atmosphere for atime of 15 minutes to several days. The reaction of the steps c) and e)is also achieved with elemental metal or low-valent metal salt such asZn, Fe, and SnCl₂ in a suitable solvent such as aqueous hydrochloric aidand methanolic hydrochloric acid at the temperature 0 to 100° C. for aperiod of 15 minutes to several days.

More preferably, the present invention relates to a process for thepreparation of a compound of the formula [2A],

which comprises

a′) ozonolysis of a compound of the formula [4A] or its salt,

in order to obtain compound of the formula [5A],

b′) cyclizing the compound of the formula [5A] with n-pentylamine inorder to obtain compound of the formula [6A],

and

c′) hydrogenating the nitro group of compound of the formula [6A] inorder to obtain the compound of the formula [2A]

Alternatively, the compound of the formula [2A] can be prepared from acompound of the formula [5A] after ozonolysis of step a′) according toclaim 7, which comprises,

d′) hydrogenating the nitro group of compound of the formula [5A],

in order to obtain the compound of the formula [7A],

and

e′) cyclizing compound of the formula [7A] with n-pentylamine in orderto obtain the compound of the formula [2A].

The present invention further relates to compounds of the formula [2],

wherein,

-   R¹ and R² are independently hydrogen, halogen, (C₁-C₅)-alkyl or    (C₁-C₅)-alkoxy;-   R³ is hydrogen;    -   (C₁-C₅)-alkyl optionally substituted with one to three moieties        independently selected from the group consisting of        (C₁-C₅)-alkoxy, hydroxy, halogen, amino, (C₃-C₇)-cycloalkyl,        heterocyclic ring or aryl in which the aryl ring is optionally        substituted with one to three moieties independently selected        from the group consisting of hydroxy, (C₁-C₅)-alkoxy, amino,        mono-(C₁-C₅)-alkylamino or di-(C₁-C₅)-alkylamino;-   R⁴ is hydrogen;    -   (C₁-C₁₀)-alkyl, optionally substituted with one to three        moieties independently selected from the group consisting of        (C₁-C₅)-alkoxy, hydroxy, halogen, amino,        mono-(C₁-C₅)-alkylamino, di-(C₁-C₅)-alkylamino and        (C₃-C₇)-cycloalkyl, a heterocyclic ring or aryl in which the        aryl ring is optionally substituted with one to three moieties        independently selected from the group consisting of hydroxy,        alkoxy, halogen, amino, mono-(C₁-C₅)-alkylamino and        di-(C₁-C₅)-alkylamino; and-   R⁵ is —OH or —O—(C₁-C₅)-alkyl.

Most preferably, said compound is5-amino-4-hydroxy-3-pentyl-3,4-dihydroquinazoline [2a].

Further the present invention concerns also a process for thepreparation of compounds of the formula [3], which comprises

halogenating a compound of the formula [8],

in order to obtain compound of the formula [3],

wherein R⁶ is chlorine or bromine.

The halogenation for the above step is preferably performed with ahalogenating reagent in a suitable solvent. Suitable hologenatingreagents are sulfuryl chloride, N-chlorosuccimide, t-butyl hypochloride,bromine, N-bromosuccimide as like. Suitable solvents are preferablydichloromethane, chloroform, acetic acid, formic acid, andtetrahydrofuran as like. Typically the reaction is conducted at thetemperature −30 to 50° C., more preferably at the temperature 0° C. toroom temperature.

The invention also relates to the compounds of the formula [3],

wherein R⁶ is chlorine or bromine.

Most preferably, the compound of formula [3] is(4S)-6-chloro-4-ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]-indolizine-3,6,10(4H)-trione[3a].

Another embodiment of the present invention concerns a process for thepreparation of a compound of the formula [1] which comprises condensinga compound of the formula [2] prepared by the process as defined abovewith a compound of the formula [3] prepared by the process as definedabove.

The hexacyclic compounds of the present invention are effective atinhibiting or preventing the growth of tumors in premalignant andmalignant cells and are useful for the treatment of carcinomas formingsolid tumors, especially of colorectal cancer, lung cancer, breastcancer, stomach cancer, cervical cancer and bladder cancer. Thehexacyclic compounds of the present invention can be used to treat suchtumors, to retard the development of such tumors, and to prevent theincrease in number of tumors.

The anti-cancer therapeutic activity of the hexacyclic compounds of thisinvention may be demonstrated by various standard in vitro assays. Suchassays described below and in the examples are known to indicateanticancer activity and are assays for cancer therapeutics. Thehexacyclic compounds of the present invention have the structuredepicted in the formula [1], and anticancer activity as determined byany standard assay, especially assays for apoptosis. The hexacycliccompounds of this invention are particularly effective to induceapoptosis in carcinoma cells, causing the death of the cell. Thus thehexacyclic compounds of this invention are the desired activities if thecompounds cause carcinoma cells to die when the cells are exposed to thehexacyclic compounds of this invention. Carcinoma cells for assays (forexample breast, lung, colorectal, etc.) are readily obtained from cellsdepositories such as the American Type Culture Collection (ATCC) or maybe isolated by skilled persons from cancer patients. The type of canceragainst which the hexacyclic compounds of this invention are most activeis determined by the type of cells used in the assays.

Carcinoma cells, grown in culture, may be incubated with a specificcompound and changes in cells viability may be determined for example,by dyes which selectively stain dead cells or by optical density (O.D.)measurement. If more than 10% of cells have died, the compound is activein inducing apoptosis. The compounds may not directly kill the cells(cellular toxicity) but may modulate certain intra- or extracellularevents which result in apoptosis. The anticancer activity of thecompounds of this invention may also be determined by assays that accessthe effects of compounds on cells growth and differentiation. Cellgrowth inhibition may be determined by adding the compound in questionto carcinoma cells in culture with dyes or radioactive precursors, anddetermining by microscopic cell counting, scintillation counting, orO.D. measurement whether the number of cells has increased over theincubation period. If the number of cells has not increased, growth hasbeen inhibited and the compound is regarded as having therapeuticactivity. Similarly, the proportion of cells which have becomedifferentiated after addition of a test compound may be determined byknown methods (i.e. measuring oxidative burst in HL-60 cells, anindicator of differentiation, by NBT(nitroblue tetrazolium). If 10% ormore cells have differentiated, then the compound is regarded as havingtherapeutic activity.

The Antiproliferative activity assay was carried out as follows. Asingle suspension of tumor cells was inoculated to the serially diluted96-well microtestplate. Then the testplate was incubated in the 5% CO₂ambience at 37° C. for 4 days (2-3×10³ cells/well). The degree of cellgrowth in a monolayer was measured by using WST-8 (Dojindo, Japan). IC₅₀values of drugs against tumor cells were calculated as the concentrationof drug yielding 50% OD of the control growth. The IC₅₀ value measuresthe drug concentration for 50% of the growth of tumor cells in vitro ascompared to the control. The results are shown in the following Table 1.

Anti-tumor activities of hexacyclic compounds of formula [1] against invitro growth of human tumor cell lines, HCT116 and DLD-1 of colorectalcancer (CRC) and QG56 and NCI-H460 of non small cell lung cancer (NSCLC)are shown in Table 1. These cell lines are commercially available viathe American Type Culture Collection (ATTC).

In Table 1,

Compound A denotes (9S)-9-ethyl-9-hydroxy-1-pentyl-1H,12H-pyrano[3″,4″:6′,7′]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,15H)-dione.Compound B denotes (9S)-9-ethyl-9-hydroxy-2-methyl-1-pentyl-1H,12H-pyrano[3″,4″:6′,7′]indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,15H)-dione.Compound C denotes(9S)-9-ethyl-9-hydroxy-2-hydroxymethyl-1-(3-methylbutyl)-1H,12H-pyrano[3″,4″:6′,7′]indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,15H)-dione.SN-38 denotes 7-ethyl-10-hydroxycamptothecin.

TABLE 1 Anti-proliferative activity assay (IC₅₀ in nM) HCT116 DLD-1 QG56NCI-H460 Compound (CRC) (CRC) (NSCLC) (NSCLC) Compound A 6.1 23 7.7 7.0Compound B 4.5 15 15 7.9 Compound C 3.1 12 7.4 4.8 SN-38 6.9 53 27 21(Reference)

For clinical use, the hexacyclic compounds of the formula [1], theirprodrugs, or salt forms thereof and the like can be administered alone,but will generally be administered in pharmaceutical admixtureformulated as appropriate to the particular use and purpose desired, bymixing excipient, binding agent, lubricant, disintegrating agent,coating material, emulsifier, suspending agent, solvent, stabilizer,absorption enhancer and/or ointment base. The admixture can be used fororal, injectable, rectal or topical administration.

In more detail, as mentioned earlier, pharmaceutical compositionscontaining a compound of the formula [1] or its prodrug are also anaspect of the present invention, as is a process for the manufacture ofsuch medicaments, whose process comprises bringing one or more compoundsof the formula [1] and, if desired, one or more other therapeuticallyvaluable substances into a galenical administration form.

The pharmaceutical compositions may be administered orally, for examplein the form of tablets, coated tablets, dragées, hard or soft gelatinecapsules, solutions, emulsions or suspensions. Administration can alsobe carried out rectally, for example using suppositories; locally orpercutaneously, for example using ointments, creams, gels or solutions;or parenterally, for example using injectable solutions.

For the preparation of tablets, coated tablets, dragées or hard gelatinecapsules, the hexacyclic compounds of the present invention may beadmixed with pharmaceutically inert, inorganic or organic excipients(pharmaceutically acceptable carriers). Examples of suitable excipientsfor tablets, dragées or hard gelatine capsules include lactose, maizestarch or derivatives thereof, talc or stearic acid or salts thereof.Suitable excipients for use with soft gelatine capsules include, forexample, vegetable oils, waxes, fats, semi-solid or liquid polyols etc.;according to the nature of the active ingredients it may however be thecase that no excipient is needed at all for soft gelatine capsules. Forthe preparation of solutions and syrups, excipients which may be usedinclude for example water, polyols, saccharose, invert sugar andglucose. For injectable solutions, excipients which may be used includefor example water, alcohols, polyols, glycerine, and vegetable oils. Forsuppositories, and local or percutaneous application, excipients whichmay be used include for example natural or hardened oils, waxes, fatsand semi-solid or liquid polyols.

The pharmaceutical compositions may also contain preserving agents,solubilizing agents, stabilizing agents, wetting agents, emulsifiers,sweeteners, colorants, odorants, salts for the variation of osmoticpressure, buffers, coating agents or antioxidants. They may also containother therapeutically valuable agents.

In summary, a pharmaceutical formulation for oral administration may begranule, tablet, sugar coated tablet, capsule, pill, suspension oremulsion, which for parenteral injection, for example, intravenously,intramuscularly or subcutaneously, may be used in the form of a sterileaqueous solution which may contain other substances, for example, saltsor glucose to make the solution isotonic. The anti-tumor agent can alsobe administered in the form of a suppository or pessary, or they may beapplied topically in the form of a lotion, solution, cream, ointment ordusting powder.

The daily dosage level of the hexacyclic compounds of the presentinvention is from 5 to 2,000 mg/m² when administered by either the oralor parenteral route. Thus tablets or capsules can contain from 5 mg to1,000 mg of active compound for single administration or two or more ata time as appropriate. In any event the actual dosage can be weight andresponse of the particular patient.

The following examples illustrate the preferred methods for thepreparation of the hexacyclic compounds of the present invention, whichare not intended to limit the scope of the invention thereto.

EXAMPLES 1. Preparation of N-(2-formyl-3-nitrophenyl)formamide [5a]

A stirred solution of 4-nitro-1H-indole (4a, 93.8 g, 0.56 mol) inDMF-MeOH (1:2, 2.7 L) cooled in a dry ice-acetone bath was bubbled withozone (generated from oxygen gas, 22 g/m³, flow rate 500 L/hr, ozonegenerator: Tokyo Keiso, 11 g/hr) for 3.5 h. The mixture was kept cool inthe dry ice-acetone bath for 1 h without stirring. The precipitategenerated was filtered and washed with H₂O to giveN-(2-formyl-3-nitrophenyl)-formamide (5a, 62.0 g, 55%); ¹H NMR (270 MHz)δ (DMSO-d₆) 7.82 (t, J=7.9 Hz, 1H), 7.91 (d, J=7.9 Hz, 1H), 8.15 (m,1H), 8.37 (br, 1H), 10.07 (s, 1H), 10.71 (br, 1H); MS (EI) m/z 194 (M⁺).

2. Preparation of 4-hydroxy-5-nitro-3-pentyl-3,4-dihydroquinazoline [6a]

N-(2-Formyl-3-nitrophenyl)formamide (5a, 20.0 g, 103 mmol) andn-pentylamine (23.9 ml, 206 mmol) were dissolved in EtOH (240 ml) andthe mixture stirred at room temperature for 3 h. After the mixture wasconcentrated under reduced pressure, EtOAc (100 ml) was added to theresidue, and the generating yellow crystal was collected by filtration.The crystal was washed with EtOAc and dried under vacuum to obtain4-hydroxy-5-nitro-3-pentyl-3,4-dihydroquinazoline (6a, 19.6 g, 72%); ¹HNMR (270 MHz) δ (CDCl₃) 0.91 (t, J=6.7 Hz, 3H), 1.26-1.38 (m, 4H),1.67-1.78 (m, 2H), 3.21 (dt, J=13.9 and 7.9 Hz, 1H), 3.77 (ddd, J=6.3,7.6 and 13.9 Hz, 1H), 6.57 (s, 1H), 6.90 (s, 1H), 7.30-7.41 (m, 2H),7.80 (dd, J=1.7 and 7.6 hz, 1H); MS (ES) m/z 264 (M⁺+1).

3. Preparation of N-(3-amino-2-formylphenyl)formamide [7a]

N-(2-Formyl-3-nitrophenyl)formamide (5a, 51.6 g, 0.27 mol) was dissolvedin THF (1 L) and 20 wt % Pd(OH)₂ on activated charcoal (4.96 g) wasadded. Hydrogenation was carried out at 37° C. in an oil bath under H₂atmosphere. After being stirred for 4 h, an additional 20 wt % Pd(OH)₂on activated charcoal (2.01 g) was added and the hydrogenation wascontinued further at 37° C. in an oil bath. After 22 h, the mixture wasfiltered and the Pd catalyst separated was washed with THF. The combinedfiltrate and washing were concentrated under reduced pressure. Thegenerated crude solid was suspended in H₂O (500 mL) and then the orangepowder was collected by filtration, washed with H₂O, and dried undervacuum to obtain N-(3-amino-2-formylphenyl)-formamide (7a, 40.2 g, 92%);¹H NMR (270 MHz) δ (CD₃OD): rotamer A (major)*6.53 (d, J=7.9 Hz, 1H),6.66 (d, J=7.9 Hz, 1H), 7.17 (t, J=7.9 Hz, 1H), 8.23 (s, 1H), 9.97 (s,1H); rotamer B (minor)*6.33 (d, J=7.9 Hz, 1H), 6.54 (d, J=7.9 Hz, 1H),7.17 (t, J=7.9 Hz, 1H), 8.40 (s, 1H), 10.05 (s, 1H). The ratio of tworotamers A and B is approximately 2:1.

¹H NMR (400 MHz) δ (DMSO-d₆, 120° C.); 6.60 (d, J=8.0 Hz, 1H), 6.70 (m,1H), 6.97 (br, 2H), 7.24 (t, J=8.0 Hz, 1H), 8.41 (s, 1H), 10.10 (s, 2H);MS (EI) m/z 164 (M⁺).

4. Preparation of 5-amino-4-hydroxy-3-pentyl-3,4-dihydroquinazoline [2a]from [6a]

4-hydroxy-5-nitro-3-pentyl-3,4-dihydroquinazoline (6a, 10.0 g, 38.0mmol) was dissolved in EtOAc-MeOH mixed solvent (2:1, 150 ml) and 3%Pt—C (1.0 g) was added. Hydrogenation was carried out at roomtemperature under H₂ atmosphere using a balloon. After 2 h, the reactionwas stopped and the mixture was filtered. The filtrated was concentratedunder reduced pressure and the obtaining residual oil was purified by ashort silica gel (100 g) column chromatography (eluent: EtOAc/MeOH=20/1)to give 5-amino-4-hydroxy-3-pentyl-3,4-dihydroquinazoline (2a, 6.56 g,74%); ¹H NMR (270 MHz) δ (CD₃OD) 0.88 (t, J=6.9 Hz, 3H), 1.23-1.41 (m,4H), 1.63-1.74 (m, 2H), 3.26-3.35 (m, 1H), 3.51-3.62 (m, 1H), 6.15 (s,1H), 6.47-6.51 (m, 1H), 6.55 (dd, J=1.0 and 7.9 Hz, 1H), 7.06 (t, J=7.9Hz, 1H), 7.43 (s, 1H); MS (ES) m/z 234 (M⁺).

5. Preparation of 5-amino-4-hydroxy-3-pentyl-3,4-dihydroquinazoline [2a]from [7a]

N-(3-amino-2-formylphenyl)formamide (7a, 40.1 g, 0.24 mol) andn-pentylamine (34.0 ml, 0.29 mol) were dissolved in EtOH (830 mL) andthe mixture was heated to reflux for 6 h in an oil bath. After beingcooled to room temperature, the mixture was concentrated under reducedpressure. The obtained residual oil was purified by silica gel columnchromatography (eluent: EtOAc only and then EtOAc/MeOH=20/1) to give5-amino-4-hydroxy-3-pentyl-3,4-dihydroquinazoline (2a, 48.8 g, 81%).

6. Preparation of(4S)-6-chloro-4-ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]-indolizine-3,6,10(4H)-trione[3a]

To a solution of(S)-4-ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione(8, 40.7 g, 0.155 mol) in dichloromethane (750 ml) was added sulfurylchloride (15.0 ml, 187 mmol) dropwise over 15 min at room temperature.The mixture was stirred at room temperature for 14 hr, and theprecipitate generated was collected by filtration. The solid wassuspended in dichloromethane (300 ml) and the solid was collected byfiltration followed by drying under vacuum to give the first crop ofpure(4S)-6-chloro-4-ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione(3a, 21.4 g, 46%) as an off-white solid. The filtrates were combined andconcentrated under reduced pressure to give a residual solid, which wassuspended in dichloromethane (200 ml). The solid was collected byfiltration and then washed in dichloromethane (200 ml). The solid wascollected by filtration and dried under vacuum to obtain the product(3a, 9.3 g, 20%) as an off-white solid; ¹H NMR (270 MHz, CDCl₃) δ0.90-1.04 (m, 3H), 1.72-1.91 (m, 2H), 3.69 (s, 1H), 4.31-4.42 (m, 1H),4.66-4.83 (m, 2H), 5.24 (d, J=17.3 Hz, 1H), 5.67 (d, J=17.3 Hz, 1H),7.35 (m, 1H).

NMR spectrum in DMSO-d₆ showed the existence of tautomeric mixture ofketo/enol forms (1:2).

¹H NMR (270 MHz, DMSO-d₆) δ Enol form: 0.68-0.89 (m, 3H), 1.65-1.90 (m,2H), 4.59 (s, 2H), 5.31 (s, 2H), 6.36 (br, 1H), 6.75 (s, 1H), 10.99 (s,1H);

Keto form: 0.68-0.89 (m, 3H), 1.65-1.90 (m, 2H), 4.06-4.18 (m, 1H),4.63-4.73 (m, 1H), 5.12-5.19 (m, 1H), 5.30-5.50 (m, 2H), 6.56 (br, 1H),7.00 (s, 1H).

7. Preparation of (9S)-9-ethyl-9-hydroxy-1-pentyl-1H,12H-pyrano[3″,4″:6′,7″]-indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10.13(9H,15H)-dione [1a]

(4S)-6-Chloro-4-ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione(3a, 17.69 g, 59.4 mmol) and5-amino-4-hydroxy-3-pentyl-3,4-dihydroquinazoline (2a, 13.86 g, 56.0mmol) were combined in acetic acid (300 mL), and the mixture was heatedat 90° C. for 2.5 h in an oil bath. After cooling to room temperature,the mixture was concentrated under reduced pressure. Water (350 mL) wasadded to the residue and the generating solid was collected byfiltration, washed with ethanol-water (1:3, 120 ml) and dried underreduced pressure to give (9S)-9-ethyl-9-hydroxy-1-pentyl-1H,12H-pyrano[3″,4″:6′,7′]indolizino[1′,2′:6,5]pyrido[4,3,2-de]quinazoline-10,13(9H,15H)-dione (1a, 22.65 g, 88%) as a brownish solid.

¹H NMR (270 MHz, DMSO-d₆)) δ 0.85-0.92 (m, 6H), 1.35-1.38 (m, 4H),1.75-1.93 (m, 4H), 3.89-3.94 (m, 2H), 5.29 (s, 2H), 5.40 (s, 2H), 6.46(s, 1H), 6.99 (dd, J=1.0, 7.4 Hz, 1H), 7.18 (s, 1H), 7.47 (dd, J=1.0,8.6 Hz, 1H), 7.62 (dd, J=7.4, 8.6 Hz, 1H), 7.86 (s, 1H); MS (ES) m/z 459(M⁺+1).

1. A compound of formula [2]:

or a salt thereof; wherein: R¹ and R² are independently hydrogen,halogen, (C₁-C₅)-alkyl or (C₁-C₅)-alkoxy; R³ is hydrogen; or(C₁-C₅)-alkyl optionally substituted with one to three moietiesindependently selected from the group consisting of (C₁-C₅)-alkoxy,hydroxy, halogen, amino, (C₃-C₇)-cycloalkyl, heterocyclic ring or andaryl in which the aryl ring is optionally substituted with one to threemoieties independently selected from the group consisting of hydroxy,(C₁-C₅)-alkoxy, amino, mono-(C₁-C₅)-alkylamino anddi-(C₁-C₅)-alkylamino; R⁴ is hydrogen; or (C₁-C₁₀)-alkyl, optionallysubstituted with one to three moieties independently selected from thegroup consisting of (C₁-C₅)-alkoxy, hydroxy, halogen, amino,mono-(C₁-C₅)-alkylamino, di-(C₁-C₅)-alkylamino, (C₃-C₇)-cycloalkyl, aheterocyclic ring and aryl in which the aryl ring is optionallysubstituted with one to three moieties independently selected from thegroup consisting of hydroxy, alkoxy, halogen, amino,mono-(C₁-C₅)-alkylamino and di-(C₁-C₅)-alkylamino; and R⁵ is —OH or—O—(C₁-C₅)-alkyl.
 2. A compound according to claim 1, which is acompound of formula [2A]:

or a salt thereof.
 3. A process of preparing a compound of claim 1, or asalt thereof, comprising: a) ozonolysis of a compound of formula [4]:

or a salt thereof, in order to obtain a compound of formula [5]:

or a salt thereof; b) cyclizing the compound of formula [5] with anamine R⁴—NH₂ to obtain a compound of formula [6]:

or a salt thereof; and c) hydrogenating the nitro group of the compoundof formula [6], or a salt thereof, in order to obtain a compound offormula [2]:

or a salt thereof; wherein: R¹ and R² are independently hydrogen,halogen, (C₁-C₅)-alkyl or (C₁-C₅)-alkoxy; R³ is hydrogen; or(C₁-C₅)-alkyl optionally substituted with one to three moietiesindependently selected from the group consisting of (C₁-C₅)-alkoxy,hydroxy, halogen, amino, (C₃-C₇)-cycloalkyl, heterocyclic ring and arylin which the aryl ring is optionally substituted with one to threemoieties independently selected from the group consisting of hydroxy,(C₁-C₅)-alkoxy, amino, mono-(C₁-C₅)-alkylamino anddi-(C₁-C₅)-alkylamino; R⁴ is hydrogen; or (C₁-C₁₀)-alkyl, optionallysubstituted with one to three moieties independently selected from thegroup consisting of (C₁-C₅)-alkoxy, hydroxy, halogen, amino,mono-(C₁-C₅)-alkylamino, di-(C₁-C₅)-alkylamino, (C₃-C₇)-cycloalkyl, aheterocyclic ring and aryl in which the aryl ring is optionallysubstituted with one to three moieties independently selected from thegroup consisting of hydroxy, alkoxy, halogen, amino,mono-(C₁-C₅)-alkylamino and di-(C₁-C₅)-alkylamino; and R⁵ is —OH or—O—(C₁-C₅)-alkyl.
 4. A process of preparing a compound of claim 2, or asalt thereof, wherein said process comprises: a′) ozonolysis of acompound of formula [4A]:

or a salt thereof, in order to obtain a compound of formula [5A]:

or a salt thereof; b′) cyclizing the compound of formula [5A], or a saltthereof, with n-pentylamine in order to obtain a compound of formula[6A]:

or a salt thereof; and c′) hydrogenating the nitro group of the compoundof formula [6A], or a salt thereof, in order to obtain a compound offormula [2A]:

or a salt thereof.
 5. A process of preparing a compound of claim 1, or asalt thereof, comprising: a) ozonolysis of a compound of formula [4]:

or a salt thereof, in order to obtain a compound of formula [5]:

or a salt thereof, d) hydrogenating the nitro group of the compound offormula [5], or a salt thereof, in order to obtain a compound of formula[7]:

or a salt thereof, e) cyclizing the compound of formula [7], or a saltthereof, with an amine R⁴—NH₂ in order to obtain a compound of formula[2]:

or a salt thereof; wherein: R¹ and R² are independently hydrogen,halogen, (C₁-C₅)-alkyl or (C₁-C₅)-alkoxy; R³ is hydrogen; or(C₁-C₅)-alkyl optionally substituted with one to three moietiesindependently selected from the group consisting of (C₁-C₅)-alkoxy,hydroxy, halogen, amino, (C₃-C₇)-cycloalkyl, heterocyclic ring and arylin which the aryl ring is optionally substituted with one to threemoieties independently selected from the group consisting of hydroxy,(C₁-C₅)-alkoxy, amino, mono-(C₁-C₅)-alkylamino anddi-(C₁-C₅)-alkylamino; R⁴ is hydrogen; or (C₁-C₁₀)-alkyl, optionallysubstituted with one to three moieties independently selected from thegroup consisting of (C₁-C₅)-alkoxy, hydroxy, halogen, amino,mono-(C₁-C₅)-alkylamino, di-(C₁-C₅)-alkylamino, (C₃-C₇)-cycloalkyl, aheterocyclic ring and aryl in which the aryl ring is optionallysubstituted with one to three moieties independently selected from thegroup consisting of hydroxy, alkoxy, halogen, amino,mono-(C₁-C₅)-alkylamino and di-(C₁-C₅)-alkylamino; and R⁵ is —OH or—O—(C₁-C₅)-alkyl.
 6. A process of preparing a compound of claim 2, orsalt thereof, wherein said process comprises: a′) ozonolysis of acompound of formula [4A]:

or a salt thereof, in order to obtain a compound of the formula [5A]:

or a salt thereof; d′) hydrogenating the nitro group of the compound offormula [5A], or a salt thereof, in order to obtain a compound offormula [7A]:

or a salt thereof, and e′) cyclizing the compound of formula [7A], or asalt thereof, with n-pentylamine in order to obtain a compound offormula [2A], or a salt thereof.
 7. A compound according to claim 1, ora salt thereof, wherein R³ is hydrogen, methyl, ethyl, propyl,hydroxymethyl, aminomethyl, chloromethyl, or trifluoromethyl.
 8. Acompound according to claim 1, or a salt thereof, wherein R⁴ is methyl,ethyl, n-propyl, 1-methylethyl, n-butyl, 1,1-dimethylethyl,2-methylpropyl, 2,2-dimethylpropyl, n-pentyl, 3-methylbutyl, 2-n-hexyl,3,3-dimethylbutyl, n-heptyl, n-octyl, benzyl, phenethyl,2-(dimethylamino)ethyl, 2-(4-morpholino)ethyl, 3-(dimethylamino)propyl,2-(pyridin-2-yl)ethyl, 2-(pyridin-3-yl)ethyl, 2-(4-methoxyphenyl)ethyl,2-(4-chlorophenyl)ethyl, 2-(4-fluorophenyl)ethyl, or 3-phenylpropyl. 9.A compound according to claim 1, or a salt thereof, wherein: R¹ ishydrogen; and R² is hydrogen or (C₁-C₃)-alkyl; R³ is hydrogen; or(C₁-C₅)-alkyl optionally substituted with one to three moietiesindependently selected from the group consisting of (C₁-C₅)-alkoxy,hydroxy, halogen, amino, (C₃-C₇)-cycloalkyl, a heterocyclic ring andaryl; R⁴ is hydrogen; or (C₁-C₈)-alkyl which is optionally substitutedwith one to three moieties independently selected from the groupconsisting of (C₁-C₃)-alkoxy, hydroxy, halogen, amino,mono-(C₁-C₃)-alkylamino, di-(C₁-C₃)-alkylamino, (C₃-C₇)-cycloalkyl, aheterocyclic ring and aryl in which the aryl ring is optionallysubstituted with one to three moieties independently selected from thegroup consisting of hydroxy, alkoxy and halogen; and R⁵ is —OH or—O—(C₁-C₅)-alkyl.
 10. A compound according to claim 1, or a saltthereof, wherein: R¹ and R² are hydrogen; R³ is hydrogen, methyl, ethyl,propyl, hydroxymethyl, aminomethyl, chloromethyl, or trifluoromethyl;and R⁴ is methyl, ethyl, n-propyl, 1-methylethyl, n-butyl,1,1-dimethylethyl, 2-methylpropyl, 2,2-dimethylpropyl, n-pentyl,3-methylbutyl, 2-n-hexyl, 3,3-dimethylbutyl, n-heptyl, n-octyl, benzyl,phenethyl, 2-(dimethylamino)ethyl, 2-(4-morpholino)ethyl,3-(dimethylamino)propyl, 2-(pyridin-2-yl)ethyl, 2-(pyridin-3-yl)ethyl,2-(4-methoxyphenyl)ethyl, 2-(4-chlorophenyl)ethyl,2-(4-fluorophenyl)ethyl, or 3-phenylpropyl; and R⁵ is —OH or—O—(C₁-C₅)-alkyl.